Desorption of plasmid DNA from anion exchangers: Salt concentration at elution is independent of plasmid size and load

Detailed studies on the sorption behavior of plasmids on anion exchangers are rare compared to proteins. In this study, we systematically compare the elution behavior of plasmid DNA on three common anion exchange resins using linear gradient and isocratic elution experiments. Two plasmids of different lengths, 8 and 20 kbp, were studied and their elution characteristics were compared to a green fluorescent protein. Using established methods for determining retention characteristics of biomolecules in ion exchange chromatography lead to remarkable results. In contrast to the green fluorescent protein, plasmid DNA consistently elutes at one characteristic salt concentration in linear gradient elution. This salt concentration was the same independent of plasmid size but differed slightly for different resins. The behavior is consistent also at preparative loadings of plasmid DNA. Thus, only a single linear gradient elution experiment is sufficient to design elution in a process scale capture step. At isocratic elution conditions, plasmid DNA elutes only above this characteristic concentration. Even at slightly lower concentrations most plasmids remain tightly bound. We hypothesize, that the desorption is accompanied by a conformational change leading to a reduced number of available negative charges for binding. This explanation is supported by structural analysis before and after elution.     

High-performance liquid chromatography micro-fraction bioactive evaluation combined with countercurrent chromatographic separation of antioxidants from Citrus peel and their tyrosinase inhibition activities

In the present study, high-performance liquid chromatography micro-fraction bioactive evaluation and high speed countercurrent chromatography were performed on screening, identification and isolation of antioxidants from Citrus peel. Three compounds were screened as antioxidants and tyrosinase inhibitors using 2,2′-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) radical cation scavenging assay and tyrosinase activity test, then they were identified as eriocitrin, narirutin and hesperidin. Moreover, the solvent system ethyl acetate-n-butanol-water (6:4:10, v/v/v) was used for separation of ethyl acetate extract of Citrus peel by high speed countercurrent chromatography. In total, 0.45 mg of eriocitrin with 87.10% purity, 2.04 mg of narirutin with 95.19% purity and 1.35 mg of hesperidin with 95.19% purity were obtained from 20 mg of ethyl acetate extract of Citrus peel in a single run and then each component was subjected to 2,2′-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) radical cation scavenging assay and tyrosinase inhibition assay. Eriocitrin showed great antioxidant activity (the half-maximum concentration: 3.65 µM) and tyrosinase inhibition activity (the half-maximum concentration: 115.67 µM), while narirutin and hesperidin exhibited moderate activity. Tyrosinase inhibition activity for eriocitrin in vitro was reported for the first time. Furthermore, molecular docking between eriocitrin and mushroom tyrosinase was also studied.     

Determination of carbonate content in barite ore by headspace gas chromatography

This paper reports a method for determining the carbonate content in barite ore using headspace gas chromatography. Based on the acidification reaction, the carbonate in the barite ore was converted to CO₂ in a closed headspace vial. When the carbonate content was significant, the pressure caused changes in the CO₂ and O₂ signals and affected the measurement accuracy. It was found that carbonate content is proportional to the intensity ratio of the CO₂ to O₂ signals. Thus, the carbonate content in barite ore can be measured indirectly using a theoretical model. The results showed that the carbonate in 3 g of barite ore sample with a particle size of 74 μm could react completely with a hydrochloric acid solution (2 mol/L) at 65°C for 5 min. The method described herein had good precision (relative standard deviation < 4.14%) and accuracy (relative differences < 6.12%). Further, the limit of quantification was 0.07 mol/L. Owing to its simplicity and speed, this method can be used for the batch determination of carbonate content in barite ore.     

Homochiral organic molecular cage RCC3-R-modified silica as a new multimodal and multifunctional stationary phase for high-performance liquid chromatography

In this work, homochiral reduced imine cage was covalently bonded to the surface of the silica to prepare a novel high-performance liquid chromatography stationary phase, which was applied for the multiple separation modes such as normal phase, reversed-phase, ion exchange, and hydrophilic interaction chromatography. The successful preparation of the homochiral reduced imine cage bonded silica stationary phase was confirmed by performing a series of methods including X-ray photoelectron spectroscopy, thermogravimetric analysis, and infrared spectroscopy. From the extracted results of the chiral resolution in normal phase and reversed-phase modes, it was demonstrated that seven chiral compounds were successfully separated, among which the resolution of 1-phenylethanol reached the value of 3.97. Moreover, the multifunctional chromatographic performance of the new molecular cage stationary phase was systematically investigated in the modes of reversed-phase, ion exchange, and hydrophilic interaction chromatography for the separation and analysis of a total of 59 compounds in eight classes. This work demonstrated that the homochiral reduced imine cage not only achieved multiseparation modes and multiseparation functions performance with high stability, but also expanded the application of the organic molecular cage in the field of liquid chromatography.     

Countercurrent chromatography separation of vitamin E isomers in a co-current mode

Vitamin E represents a group of lipophilic phenolic compounds, including α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol, and α-tocotrienol, β-tocotrienol, γ-tocotrienol, and δ-tocotrienol isomers. Different forms of vitamin E have been proven to exhibit varying biological activities. However, due to their structural similarities, the separation of vitamin E isomers is a challenging task. Therefore, it is crucial to establish an efficient method for isolating individual isomers. In this study, co-current countercurrent chromatography was employed to isolate vitamin E isomers from commercial capsules using a n-heptane-methanol-water (10:9.5:0.5, v/v) solvent system. The partition coefficients of the main constituents in the capsules ranged from 0.94 to 6.23, requiring over 450 min for a complete separation. To improve separation efficiency, a co-current elution mode was implemented and the flow rates of the two liquid phases as well as sample amount were examined. The results suggested that increasing the flow rate of the stationary phase and sample size could result in more effective separation, shorter separation time, and higher yield. It proved that co-current countercurrent chromatography was an effective method for the separation of vitamin E isomers.     

Electrophoretically deposited sulfonated poly(styrene-co-divinylbenzene) on a screw for microextraction of cationic dyes from aqueous solutions

In the present work, a novel solid-phase microextraction on a screw (MES) was employed to extract cationic dyes (malachite green, methylene blue, and rhodamine B) from food samples and fish breeding pool water. The sulfonated poly(styrene-co-divinylbenzene) was electrophoretically deposited on the surface of the grooves of a screw. Then the screw was placed inside a silicon tube as a holder to create a channel to run a test solution through it. The extracted dyes on the coated screw were eluted by a suitable eluent. High-performance liquid chromatography with an ultraviolet/visible detector was utilized for the separation and analysis of the analytes. The effective parameters of the analyte extraction efficiency were optimized. Under optimum conditions, the limits of detection were 0.15 μg/L, and calibration curves were linear in the range of 0.50–250.00 μg/L, with coefficients of determination > 0.989 for all studied dyes. The relative standard deviations of intra and inter-day (n = 3) were in the range of 2.8%–7.0% and 7.0%–9.5%, respectively. The MES was applied as a simple and repeatable method with acceptable relative recoveries (82.0%–103.0%) for the determination of cationic dyes in grape nectar, ice pop, jelly powder, and fish breeding pool water.     

Development and validation of high-performance liquid chromatography method for determination of clarithromycin in pharmaceutical tablets

Clarithromycin is a very important macrolide antibiotic used to treat bacterial infections in human and veterinary medicine. This study reports the development and validation of cost-effective, simple, precise, accurate, and robust high-performance liquid chromatography (HPLC) for the determination of clarithromycin (CLA) in tablets. Reversed-phase chromatography was conducted using a standard column at 55°C with ultraviolet detection at 215 nm. A mobile phase consisting of acetonitrile –2-methyl-2-propanol –potassium phosphate buffer was used at a flow rate of 1.0 mL/min. The proposed method displayed good linearity, precision, accuracy, robustness, and specificity. The present HPLC was compared with capillary electrophoresis and bioassay methods and the results indicated that there was no significant difference between these methods. Moreover, the obtained results demonstrated the validity of the isocratic HPLC, which allows reliable quantitation of CLA in pharmaceutical samples. Thus, it can be used as a substitute alternative methodology for the routine quality control of this medicine, in situations where other methods are less accessible in the laboratory.     

Chemical profiling of Ziziphi spinosae semen using on-line comprehensive two-dimensional liquid chromatography-mass spectrometry based on a novel phthalic anhydride bonded stationary phase

Ziziphi spinosae semen has been widely used to treat insomnia and anxiety. To profile its chemical components, an online comprehensive two-dimensional liquid chromatography-mass spectrometry was developed. In this two-dimensional liquid chromatography system, a novel phthalic anhydride-bonded stationary phase column was combined with a C18 column. As a result, this new stationary phase exhibited remarkable differences in separation selectivity from C18, achieving a good orthogonality of 83.3%. Moreover, this new stationary phase with weaker hydrophobicity than C18 realized solvent compatibility in the online configuration. Coupled with tandem MS, 154 compounds were identified, including 51 unreported compounds. Compared with one-dimensional liquid chromatography-mass spectrometry, this online two-dimensional liquid chromatography-mass spectrometry system exhibited a much higher resolving power in isomer separation. This work provided an effective separation and characterization method for the material basis of Ziziphi spinosae semen. This strategy provides ideas for the material basis research of other traditional Chinese medicines.     

Extraction and isolation of diphenylheptanes and flavonoids from Alpinia officinarum Hance using supercritical fluid extraction followed by supercritical fluid chromatography

In this paper, an off-line combination method of supercritical fluid extraction and supercritical fluid chromatography was developed for the selective extraction and isolation of diphenylheptanes and flavonoids from Alpinia officinarum Hance. The enrichment of target components was successfully achieved using supercritical fluid extraction with the following conditions (8% ethanol as co-solvent at 45°C and 30 MPa for 30 min). Taking full advantage of the complementarity of supercritical fluid chromatography stationary phases, a two-step preparative supercritical fluid chromatography strategy was constructed. The extract was firstly divided into seven fractions on a Diol column (250 × 20 mm internal diameter, 10 μm) within 8 min by gradient elution increasing from 5% to 20% modifier (methanol) at 55 ml/min and 15 MPa. Then the seven fractions were separated by using a 1-AA or a DEA column (250 × 19 mm internal diameter, 5 μm) at 50 ml/min and 13.5 MPa. This two-step strategy showed superior separation ability for structural analogs. As a result, seven compounds, including four diphenylheptanes and three flavonoids with high purity, were successfully obtained. The developed method is also helpful for the extraction and isolation of other structural analogs of traditional Chinese medicines.     

Ternary eluent compositions in supercritical fluid chromatography improved fingerprinting of therapeutic peptides

Currently, little information has been published on the application of ternary eluent compositions in supercritical fluid chromatography for separating peptides. This work investigates the benefits of adding acetonitrile to methanol as the modifier. Three cyclic antibiotic peptides (bacitracin, colistin, and daptomycin) ranging between 1000 and 2000 Da were chosen as model substances. The ternary mixture of carbon dioxide, methanol, and acetonitrile is optimized to increase the resolution of the peptide's fingerprint. In addition, varying compositions of methanol and acetonitrile were found to change the elution order of the analytes, which is a valuable tool during method development. An individual gradient method using two Torus 2-PIC columns (each 100 × 3.0 mm, 1.7 μm), carbon dioxide, and a modifier consisting of acetonitrile/methanol/water/methanesulfonic acid (60:40:2:0.1, v:v:v:v) was optimized for each of the peptides. Subsequently, a generic method development protocol applicable to polypeptides is proposed.